EP0599159B1 - Heterogeneous protein mixture with alpha-L-rhamnosidase activity, process for preparation and use. - Google Patents

Description

The invention relates to a heterogeneous protein mixture with the activity of an α-L-rhamnosidase which catalyzes the cleavage of the bond between terminal L (+) - rhamnose and aglycone of rhamnose-containing glycosides, a process for biotechnical production and their use for the production of L (+ ) Rhamnose (6-deoxy-L-mannose). This latter sugar is referred to below as L-rhamnose.

L-rhamnose is a very good chiral building block for the production of various organic compounds. L-rhamnose or its derivatives are used increasingly in the synthesis of pharmaceutical products and crop protection agents, as well as in the field of plant and animal cell cytology, microbiology, immunobiology and aroma production. For example, Produce 2,5-dimethyl-4-hydroxy-2,3-dihydrofuran-3-one (Furaneol®) with L-rhamnose as the starting compound, which in turn serves as the basic body of various flavorings in the food and fragrance industry.

L-rhamnose is very difficult to access chemically. However, it can be obtained extractively from various natural sources after acid hydrolysis, for example from the flavone glycosides hesperidin, rutin, naringin, quercitrin or, for example, from gum arabic or seaweed. [(Biotechnology and Bioengineering, Vol. 33, p. 365 (1989), RJ Linhardt et al .; EP-A-0 317 033; JPA 62293]. The costly isolation steps for L-rhamnose have a disadvantage for these processes, in part using organic solvents, furthermore the aromatic, potentially toxic waste products arising in the processing and those in the Composition of fluctuating ingredients of the natural sources depending on the seasonal rhythm.

L-rhamnose can also be fermented in the form of rhamnose-containing heteropolysaccharides with the help of bacteria of various genera such as B. Alcaligenes, Acinetobacter, Klebsiella, Streptococcus or Lactobacillus.
[Enzyme Microb. Technol., Vol. 10, p. 198 (1988), M. Graber et al .; J. Amer. Chem. Soc., Vol. 71, p. 4124 (1945), FG Jarvis and MJ Johnson; J. Bacteriol., Vol. 68, p. 645 (1954), G. Hauser and ML Karnovsky].

Disadvantages of these processes are the low yields which are usually due to viscosity and the separation of the L-rhamnose from a mixture of different sugars which is necessary after hydrolytic cleavage of the heteropolysaccharide.

Numerous publications and patents deal with the fermentative extraction of rhamnolipids by Pseudomonas aeruginosa. [Applied and Environmental Microbiology, Vol. 51, No. 5, p. 985 (1986), H.E. Reiling et al .; J. Chem. Techn. Biotechnol., Vol. 45, p. 249 (1989), K. Venkata Ramana et al .; U.S. Patent 4,933,281, Daniels et al .; German Offenlegungsschrift 2,150,375, 1972; U.S. Patent 4,814,272, Wagner et al.]

In the culture solution of microorganisms there are basically 4 rhamnolipids (RL1 - RL4, see Fig. 1), which consist of 1 or 2 L (+) - rhamnose units and one or two β-hydroxydecanoic acids [Z. Natural science. 40 c, p. 61 (1985), C. Syldatk et al.]. Rhamnolipids 1 and 3 make up the largest proportion in terms of quantity.

Figure 1: Rhamnolipids from microorganisms

It is also known to obtain L-rhamnose from flavone glycosides, rhamnolipids or oligosaccharides by enzymatic cleavage with the soluble or immobilized α-L-rhamnosidases naringinase and hesperidinase [US Pat. 5,077,206; Eur. Pat. 88202595.0; Turecek, P. and Pittner, F., Appl. Biochem. and biotech. 13, 1-13 (1986)]. Naringinase and hesperidinase each have a molecular weight of approximately 90 kd and were isolated from Penicillium decumbens and Aspergillus niger.

Naringinase and hesperidinase catalyze the cleavage of the bond between two monosaccharides, mainly the cleavage of terminal rhamnose from flavanone glycosides, e.g. from hesperidin, naringin or from rhamnolipid 3 or 4.

The catalysis of the bond cleavage between terminal rhamnose and an aglycon of rhamnose-containing glycosides by nariginase and hesperidinase is significantly slower (factor: 10-100; see Example 1).

This means that a very long period of time is necessary to completely split off the rhamnose from the rhamnolipid 1-4 mixture, since that rhamnolipid 1 and 3, which are most commonly present in the culture solution of microorganisms, consist of L-rhamnose and aglycon (fatty acid).

It has now surprisingly been made from Penicillium sp. isolated a heterogeneous protein mixture with the activity of an α-L-rhamnosidase, which catalyzes the cleavage of the bond between terminal L-rhamnose and aglycon of rhamnose-containing glycosides, that is to say the reverse specificity compared to the known α-L-rhamnosidases nariginase and hesperidinase.

• 1. ein heterogenes Proteingemisch mit der Aktivität einer α-L-Rhamnosidase, erhältlich
  • durch Fermentation von Penicillium sp. DSM 6825 und/oder 6826
  • Abtrennung der Biomasse von der Kulturbrühe und
  • Konzentrierung des Kulturüberstands.
• 2. ein heterogenes Proteingemisch mit der Aktivität einer α-L-Rhamnosidase und zwar mit einem Molekulargewicht von 60 - 100 kd und den aminoterminalen Aminosäuresequenzen oder welche die Spaltung der Bindung zwischen terminalen L-Rhamnose und Aglykon von rhamnosehaltigen Glykosiden katalysiert.
• 3. Ein Verfahren zur Herstellung eines solchen heterogenen Proteingemisches mit der Aktivität einer α-L-Rhamnosidase, dadurch gekennzeichnet, daß Penicillium sp. in einem Nährmedium kultiviert wird, wobei sich ein heterogenes Proteingemisch mit der Aktivität einer α-L-Rhamnosidase in der Kultur anhäuft, die Biomasse von der Kulturbrühe abgetrennt und der so erhaltene Kulturüberstand aufkonzentriert wird.
• 4. Verwendung eines solchen heterogenen Proteingemisches mit der Aktivität einer α-L-Rhamnosidase zur Herstellung von L-Rhamnose.
• 5. Penicillium sp. DSM 6825
• 6. Penicillium sp. DSM 6826

The invention thus relates

• 1. a heterogeneous protein mixture with the activity of an α-L-rhamnosidase available
  • by fermentation of Penicillium sp. DSM 6825 and / or 6826
  • Separation of the biomass from the culture broth and
  • Concentration of the culture supernatant.
• 2. a heterogeneous protein mixture with the activity of an α-L-rhamnosidase, specifically with a molecular weight of 60-100 kd and the amino-terminal amino acid sequences or which catalyzes the cleavage of the bond between terminal L-rhamnose and aglycon of rhamnose-containing glycosides.
• 3. A method for producing such a heterogeneous protein mixture with the activity of an α-L-rhamnosidase, characterized in that Penicillium sp. is cultivated in a nutrient medium, a heterogeneous protein mixture with the activity of an α-L-rhamnosidase accumulating in the culture, the biomass being separated from the culture broth and the culture supernatant thus obtained being concentrated.
• 4. Use of such a heterogeneous protein mixture with the activity of an α-L-rhamnosidase for the production of L-rhamnose.
• 5. Penicillium sp. DSM 6825
• 6. Penicillium sp. DSM 6826

The invention is described in detail below, particularly in its preferred embodiments.

The compound L (+) - rhamnose (= 6-deoxy-L-mannose) is referred to as L-rhamnose.

An aglycone is a compound or the part of a compound that contains no sugar. In this invention, fatty acid or flavone compounds are referred to as aglycone.

The following abbreviations are used for the amino acids, which correspond to the "single-letter code" known from the specialist literature.

The heterogeneous protein mixture with the activity of an α-L-rhamnosidase can be obtained from the culture broth both in small amounts (amounts up to 1 gram) and in large amounts (≤ 1 kg), since the process for production on a laboratory scale (fermentation of Microorganisms in volumes up to 1 liter) and on an industrial scale (fermentation of the microorganisms on a cubic meter scale) can be carried out.

The molecular weight of the heterogeneous protein mixture according to the invention with the activity of an α-L-rhamnosidase is determined using SDS gel electrophoresis (SDS = sodium dodecyl sulfate) and gel chromatography. This method of gel chromatography is described, for example, in "Molecular Biology of the Cell", Bruce Alberts et al., Garland Publishing, Inc. New York & London, 3rd edition, 1983, pp. 174, 265-266.

The abbreviation "IEP" stands for "isoelectric point" and is defined as the pH at which the net charge of the protein or in the present case of the enzyme is zero. The IEP is determined using chromatofocusing.

Penicillium sp. was isolated from a compost of garden waste in Bad Soden, Hessen, Germany. The microorganism was isolated and purified by methods known to those skilled in the art by culture dilution and plating on selective agar. For example, the compost sample can be suspended in 0.9% saline solution and an enrichment culture can be created from this suspension in a selective medium with rhamnolipids and / or rhamnolipid derivatives, preferably rhamnolipid-2-C ₁ -C ₁₈ -alkyl esters as the only carbon source. Rhamnolipid -2-C ₁ -C ₄ alkyl esters, such as rhamnolipid-2-methyl ester or rhamnolipid-2-tert-butyl ester, are particularly preferably used as the C source.

Formulas Penicillium sp. DSM 6825 and DSM 6826 have the following morphological characteristics (according to RA Samson et al., Introduction to Food-borne Fungi, Institute of the Royal Netherlands Academy of Arts and Sciences, 3rd edition, 1988): Penicillium sp. DSM 6825 Branching of the conidia monoverticilliat Phialids ampoule shape Conidia prickly Penicillium sp. DSM 6826 Branching of the conidia biverticilliat Phialids bottle-shaped Conidia spicy

Penicillium sp. DSM 6825 and 6826 can be fermented together or separately.

Instead of isolates DSM 6825 and / or 6826, mutants and variants can also be used if they produce the enzyme α-L-rhamnosidase. Such mutants can be known in a manner known per se by physical means, for example radiation, such as with ultraviolet or X-rays, or chemical mutagens, such as, for example, ethyl methanesulfonate (EMS), 2-hydroxy-4-methoxy-benzophenone (MOB) or N-methyl -N'-nitro-N-nitrosoguanidine (MNNG) are generated.

The procedure for producing the above-mentioned heterogeneous protein mixture with the activity of an α-L-rhamnosidase is as follows:

Subsequently for the isolation and purification of Penicillium sp. (see p. 6) by multiple passage of the mixed culture of the resulting microorganisms in selective medium, an enrichment of the microorganisms that produce the heterogeneous protein mixture according to the invention is achieved.

The microorganisms thus obtained are plated on agar plates (selective medium) in order to obtain pure cultures from the mixed culture.

The pure cultures are propagated and tested for their ability to form the heterogeneous protein mixture according to the invention.

It shows that microorganisms of the genus Penicillium sp. form the enzyme of the invention. The fungus genus is determined on the basis of morphological, taxonomic and biochemical criteria according to methods known to the person skilled in the art. The colony color is green.

The investigation of the ability of Penicillium sp. Colonies for the formation of the heterogeneous protein mixture according to the invention lead to the isolation of two strains which are distinguished by a particularly high production of α-L-rhamnosidase. These two strains are: Penicillium sp. DSM 6825 and DSM 6826.

The strains were obtained from the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1B, Braunschweig, Hesse, Germany, according to the rules of the Budapest Treaty on November 29, 1991 under the numbers: Penicillium sp. DSM 6825 and 6826 deposited.

The microorganisms in question are cultivated under those for Penicillium sp. usual conditions. As a result, they are grown on complex or defined media, preferably containing the media

Yeast extract, casamino acids, corn steep, meat extract, peptone, casein, gelatin, trypton, nitrate, ammonium or urea as nitrogen source and starch, dextrin, sucrose, glucose, glycerin, malt extract as carbon source.

Magnesium, calcium, sodium, potassium, iron, zinc, cobalt or phosphate can be used as further components. The use of rhamnolipids or their alkyl esters (raw mixtures or purified rhamnolipids) as a C source, alone or in combination with other C sources, has proven to be particularly preferred.

The cultivation takes place at 27 ° C for 72-120 hours. by filtration or centrifugation to separate the biomass, the α-L-rhamnosidase being largely in the supernatant.

The supernatant can be concentrated by ultrafiltration or lyophilized. Further cleaning steps such as Precipitation, anion exchange chromatography, chromatofocusing HIC chromatography (hydrophobic interaction chromatography), exclusion chromatography and affinity chromatography can be carried out depending on the desired purity of the enzyme.

The purification is preferably carried out by filtration to separate the biomass, subsequent ultrafiltration to concentrate the enzyme in the remaining supernatant, subsequent anion exchange chromatography, chromatofocusing and finally the exclusion chromatography.

The heterogeneous protein mixture with the activity of an α-L-rhamnosidase has a molecular weight of 60-100 kD depending on the degree of glycosylation and an isoelectric point of 5.6-5.8. The pH optimum of the cleavage of p-nitrophenyl-α-L-rhamnopyraside is 5.0 - 5.5, the temperature optimum at 50 - 55 ° C.

The determination of the amino terminal sequence from the strain Penicillium sp. DSM 6826 and the naringinase are generally made according to the Edman method known from the literature. For this purpose, amino acid chains are mixed with phenyl isothiocyanate under suitable conditions. The chemical compound attaches itself preferentially to the free amino terminal amino group. In the presence of anhydrous acid, the terminal amino acid is split off as a carbamyl derivative. This compound is examined to identify the amino terminal amino acid. The rest of the amino acid chain (which now lacks the original amino acid) can now be subjected to renewed treatment with phenyl isothiocyanate to determine the next amino acid, etc. In principle, this process can be carried out several times in succession, so that the total amino acid frequency of the chain can be derived. In the present case, the N-terminal amino acid sequencing is also carried out using a gas phase sequencer (Type 477 A from Applied Biosystems) and the amino acid analysis is carried out using an online amino acid analyzer (Type 130 A PTC analyzer from Applied Biosystems). The methods are published in FEBS Letters, Vol. 292, pp. 405-409, 1991.

The following amino-terminal partial sequences could be determined for the heterogeneous protein mixture with the activity of an α-L-rhamnosidase: or

The heterogeneous protein mixture according to the invention with the activity of an α-L-rhamnosidase can be used in free or immobilized form, all common immobilization methods being suitable for this. For example, silica gel can be used as a carrier (for reasons of cost).

The heterogeneous protein mixture with the activity of an α-L-rhamnosidase catalyzes the cleavage of the bond between terminal L-rhamnose and aglycone of rhamnose-containing glycosides and is therefore suitable for the production of rhamnose.

The cleavage is carried out in aqueous solutions which are buffered or unbuffered. Aqueous solutions are, for example, the culture broth of the microorganism (no buffering required) or distilled water. In the latter case, buffering with phosphate or tris buffer, preferably ammonium acetate buffer, is necessary (concentration of the buffer: 5-100 mM, preferably 10-50mM). The pH of the aqueous solution is pH 3.5-8, preferably pH 5-6. The temperature necessary for the enzyme activity is between 4 ° C-65 ° C, preferably 45 ° C-55 ° C. The reaction time depends on the amount of enzyme and substrate and also slightly on the temperature. At a temperature of 45 ° C-55 ° C, the reaction time is 2-24 hours, preferably 5-8 hours. A shorter reaction time makes sense at higher temperatures because the enzyme degrades more easily. The amount of substrate in the batch (= amount of rhamnolipids) is a maximum of 200 g / l, the amount of enzyme 0.1-50 U / g rhamnolipids, preferably 1-10 U / g and particularly preferably 5 U / g rhamnolipids.

After the reaction has ended, the L-rhamnose is isolated from the solution by separating the fat phase by means of centrifugation or decanter until phase separation has taken place, where appropriate the aqueous phase is subsequently clarified, e.g. using activated carbon. Clarification is the removal of turbidity and colorants. This step is useful if you want to get the purest possible L-Rhamnose. Finally, the aqueous solution is concentrated and the L-rhamnose is crystallized out.

example 1 Cleavage of rhamnolipids 1 and 3 by naringinase and hesperidinase

10 g rhamnolipid 1 or 3 are dissolved in 100 ml ammonium acetate buffer (50 mM, pH 5.5) or aqua bidest. emulsified and mixed with 150 U naringinase or hesperidinase (Sigma, Germany). The reaction takes place at 70 ° C. with stirring. The values for v _max achieved under these conditions are summarized in Table 1. The naringinase cleaves the 10 g of rhamnolipid 3 used in 2.5 g of rhamnose (∼ 98% yield) and rhamnolipid1 in 4 hours (hesperidinase 7 hours). The cleavage of rhamnolipid 1 is much slower (see Table 1) and incomplete, probably due to the inactivation of the enzyme over the relatively long period.

The reaction was monitored by TLC, the rhamnose was determined quantitatively using HPLC. Table 1: DC Eluent CHCl ₃ / CH ₃ OH / HAc 65: 5: 2 DC plate Silica gel 60 F254 Spray reagent MeOH / HAc / H ₂ SO ₄ conc. / Anisaldehyde 85: 10: 5: 1 development 5 minutes 120 ° C

Enzymatic cleavage of rhamnolipid 1 and 3 using hesperidinase and naringinase [The v _max values relate to 1 U α-L-rhamnosidase activity; 1 U is defined as the amount of enzyme capable of cleaving p-nitrophenyl-α-L-rhamnopyranoside per 1 µ mol / minute]: Hesperidinase (Sigma No. H-8137, Penicillium spec.) "v _max " rhamnolipid 3: _~ 50µgmn ^-1 u ^-1 "v _max " rhamnolipid 1: _~ 0.5µgmin ^-1 u ^-1 Naringinase (Sigma No. N-1385, Penicillium decumbens) "v _max " rhamnolipid 3: _~ 80µgmin ^-1 u ^-1 "v _max " rhamnolipid 1: _~ 5.0µgmin ^-1 u ^-1 HPLC pillar HPAP (100x7.8 mm) biorad Precolumn Carbo P (30x4.6) biorad temperature 85 ° C Eluent Aqua bidest. Flow 0.4 ml / minute assignment 5µl detector Differential refractometer (Beckmann)

The rhamnose is worked up according to the usual methods described in the literature (PCT-EP 91-01426).

Example 2 Cleavage of rhamnolipids 1 and 3 by the heterogeneous protein mixture with the activity of an α-L-rhamnosidase from Penicillium sp. DSM 6825 and / or 6826.

10 g of rhamnolipid 1 or 3 are emulsified in 100 ml of ammonium acetate buffer (50 mM, pH 5.0) or double-distilled water and mixed with 150 U of the enzyme according to the invention. The reaction takes place at 50 ° C. with stirring. The values for v _max achieved under these conditions are summarized in Table 2. The α-L-rhamnosidase according to the invention cleaves in about 5-8 hours (α-L-rhamnosidase from Penicillium sp. DSM 6825: about 8 hours; α-L-rhamnosidase from Penicillium sp. DSM 6826: approx. 5 hours) the 10 g of rhamnolipid 1 used in 3.05 g of rhamnose (~ 94% yield) and the corresponding fatty acid. The cleavage of rhamnolipid 3 is much slower (see Table 2) and incomplete, probably also due to the inactivation of the enzyme over a relatively long period of time.

Table 2:

Enzymatic cleavage of rhamnolipids. The v _max . Values relate to 1 U α-L-rhamnosidase activity; 1 U is defined as the amount of enzyme that is able to cleave 1 µmol of p-nitrophenyl-α-L-rhamnopyranoside per minute: α-L-rhamnosidase Penicillium sp. DSM 6825 from Penicillium sp. 6825: "v _max " rhamnolipid 3: _~ 0.03µgmin ^-1 u ^-1 "v _max " rhamnolipid 1: _~ 40.0µgmin ^-1 u ^-1 α-L-rhamnosidase Penicillium sp. DSM 6826 from Penicillium sp. 6826: "v _max " rhamnolipid 3: _~ 0.05µgmin1. ^-1 u ^-1 "v _max " rhamnolipid 1: _~ 70.0µgmin ^-1 u ^-1

The L-rhamnose is isolated again according to the known methods, and the fatty acid which is split off can also be isolated by extraction in acid.

Example 3 Screening for strains producing α-L-rhamnosidase

From various soil samples, microorganisms on nutrient media were enriched with rhamnolipid-2-methyl ester or rhamnolipid-2-tert-butyl ester as the only C source using common microbiological methods (Drews, microbiological internship, 45-84, Springer Verlag 1983) and pure cultures were isolated. Of the approximately 400 strains isolated, around 37 were able to break down rhamnolipids. However, significant α-L-rhamnosidase activity could only be demonstrated in some of the strains; two strains (Penicillium sp. DSM 6825 and Penicillium sp. DSM 6826) proved to be particularly good producers.
P-Nitrophenyl-α-L-rhamnopyranoside is used as the substrate for the detection of the α-L-rhamnosidase activity. 10 mg of this substrate are dissolved in 10 ml of ammonium acetate buffer (pH 5.5 50 mM). 900 μ l of this solution is mixed with 100 μ l culture or cell lysates and at 40 ° C incubated. After 0.3, 6, 9, 12 minutes, 200 ul are removed and mixed with 800 ul 200 mM borate buffer pH 9. The cleavage of p-nitrophenol is monitored photometrically at 410 nm, and naringinase served as a control (based on Romero et al. Anal. Biochem. 149, 566-571 (1985).

Example 4 Production of α-L-rhamnosidases - activities with the strains Penicillium sp. DSM 6825 and Penicillium sp. DSM 6826.

The strains are first spread on agar plates with HA medium (yeast extract 4 g / l, malt extract 10 g / l, glucose 4 g / l, agar 20 g / l pH 6.0) and 10-14 days at 25 ° C to incubated for good sporulation. A spore suspension (50 ml 0.9% NaCl; 0.05% Tween 80) is prepared from two well-covered plates and used to inoculate 10 l of production medium.

The following nutrient solution is used as the production medium: 3 g / l rhamnolipid 1 or 2 or alkyl esters of rhamnolipids or rhamnolipid mixtures (e.g. concentrated culture filtrate from Pseudomonas aeruginosa, 1 g / l KH ₂ PO ₄ , 0.5 g / l (NH ₄ ) ₂ SO ₄ , 0.1 g / l MgSO ₄ x 7H ₂ O, 0.1 g / l CaCl ₂ , 0.1 g / l casaminoacids pH 5.5 The culture is carried out in a 10 l leaf stirred reactor at 300 rpm, 0.6 vvm , 27 ° C. at pH 5.5 The culture time is 5-10 days.

At the end of the fermentation, the cells are separated by filtration and the culture filtrate is sterile filtered (0.22 µm). This culture filtrate contains most (over 90%) of the α-L-rhamnosidase activity (5000 U / l) and can be used directly or after lyophilization or concentration by ultrafiltration on a 10 kD membrane to cleave the rhamnolipids.

Example 5 Isolation and characterization of the α-L-rhamnosidase from Penicillium sp. DSM 6826

For the further purification of the α-L-rhamnosidase, a concentrate with an activity of 50 U / ml was assumed.

As a first step, chromatography on Sepharose Q with 20mM Tris / HCL pH 7.6 is carried out. Elution is carried out with a gradient of 0-0.5 M NaCl, the yield achieved is approximately 80% with a cleaning factor of 5; the enzyme has a spec. Activity of 62 U / mg protein.

This fraction is further chromatographed on a Mono P column (Pharmacia) (25 mM imidazole / HCl against PBE 74, pH 5.0, 1:12). The α-L-rhamnosidase is eluted at a pH of 5.6-5.8. The yield is about 70%.

After appropriate buffering, the protein is chromatographed on a Superose 12 column (1x30 cm). 100 mM ammonium acetate pH 5.0 with 100 mM NaCl is used as buffer. The examination using SDS gel electrophoresis reveals a protein band in the range of 60-100 kd depending on the degree of glycosylation of the enzyme.

The investigations summarized in the following table were carried out with the purified enzyme. Table 3 Influence of various substances on the activity of the α-L-rhamnosidase according to the invention substance % Activity control 100 CaCl ₂ (20 mM) 127 MgSO ₄ (2 mM) 106 KCl (100 mM) 69 CsCl (2 mM) 96 CoCl ₂ (2 mM) 41 CuCl ₂ (0.5 mM) 40 FeSO ₄ (0.5 mM) 100 MnCl ₂ (2 mM) 27 ZnCl ₂ (2 mM) 65 EDTA (10 mM) 22 EDTA / CaCl2 (10/10 mM) 90 EGTA (10 mM) 60 EGTA / MgSO4) (10/10 mM) 124 L-rhamnose (0.5 M) 51 L-rhamnose (1.0 M) 39.5 L-rhamnose (1.5 M) 26.4

Example 6 Determination of the N-terminal sequence of the α-L-rhamnosidase from the strain Penicillium sp. DSM 6826 and from Naringinase

The enzyme obtained according to Example 5 from Penicillium sp. DSM 6826 as well as the naringinase from Penicillium decumbens (crude enzyme, Sigma No. N-1385) purified by the same method are cleaned again using an acrylamide gel (10%), and after transfer of the polypeptides by means of electroblotting onto a ProBlot® membrane ( Applied Biosystems) (Sequencers, No. 42, April 1990, Applied Biosystems). The naringinase was obtained as a single band, while the purified enzyme from DSM 6826 could be resolved into two very closely neighboring bands.
The N-terminal sequence of the three polypeptide chains is determined by means of the Applied Biosystems peptide sequencer 477a. The results in Table 4 clearly show that both polypeptide chains of the active preparation from strain DSM 6826 are different from naringinase (Sigmu No.-N-1385). Table 4 Peptides N-terminal sequence Naringinase (96,000 D) ASVPXGEXILAPSSIELIPT α-L-rhamnosidase 6826, peptide I (96,000 D) DTNDQTSAKVDRGTFDDPAARL α-L-rhamnosidase 6826, peptide II (83 500 D) FFGSX ₁ QSLYLKLVLKFGTLFD (X ₂ ) A X ₁ probably means cysteine
X ₂ amino acid not determined

Claims (13)

1. A heterogeneous protein mixture having the activity of an α-L-rhamnosidase, which can be obtained by

   - fermenting Penicillium sp. DSM 6825 and/or 6826

   - separating off the biomass from the culture broth,

   - concentrating the culture supernatant, with the heterogeneous protein mixture having a higher specificity for the cleavage of the bond between terminal L-rhamnose and an aglycone of rhamnose-containing glycosides than for the elimination of terminal rhamnose from flavanone glycosides.
2. A heterogeneous protein mixture having the activity of an α-L-rhamnosidase, having a molecular weight of 60-100 kd and an IEP, ascertained by chromatofocussing, of 5.6-5.8, which heterogeneous protein mixture having the activity of an α-L-rhamnosidase contains the amino-terminal amino acid sequence or and catalyzes the cleavage of the bond between terminal rhamnose and the aglycone of rhamnose-containing glycosides at a higher specificity than the elimination of terminal rhamnose from flavanone glycosides.
3. The heterogeneous protein mixture having the activity of an α-L-rhamnosidase as claimed in claim 2, wherein the mixture originates from Penicillium sp.
4. The heterogeneous protein mixture having the activity of an α-L-rhamnosidase as claimed in claim 1, wherein the IEP of the enzyme, ascertained by chromatofocussing, is 5.6-6.8.
5. The heterogeneous protein mixture having the activity of an α-L-rhamnosidase as claimed in one or more of claims 2-4, wherein the mixuture originates from Penicillium sp. DSM 6826.
6. The heterogeneous protein mixture having the activity of an α-L-rhamnosidase as claimed in claim 2, wherein the aglycone is a fatty acid.
7. The heterogeneous protein mixture having the activity of an α-L-rhamnosidase as claimed in claim 1 or 2, which heterogeneous protein mixture having the activity of an α-L-rhamnosidase catalyzes the cleavage of rhamnolipids.
8. A process for preparing a heterogeneous protein mixture having the activity of an α-L-rhamnosidase as claimed in claim 1, wherein Penicillium sp. is cultivated in a nutrient medium until a heterogeneous protein mixture having the activity of an α-L-rhamnosidase accumulates in the culture, the biomass is separated off from the culture broth, and the culture supernatant thus obtained is concentrated.
9. The process as claimed in claim 8, wherein the nutrient medium contains rhamnolipids and/or rhamnolipid derivatives as the carbon source.
10. The process as claimed in claim 8, wherein Penicillium sp. DSM 6825 and/or 6826 are cultivated.
11. Use of a heterogeneous protein mixture having the activity of an α-L-rhamnosidase as claimed in claim 1 or 2 for the preparation of L-rhamnose.
12. Penicillium sp. DSM 6825.
13. Penicillium sp. DSM 6826.